Summary Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiological agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Lytic KSHV replication is crucial to tumorogenicity because in KS lesions it sustains the population of latently infected cells that would otherwise be lost by segregation of latent viral episomes as spindle cells divide. Despite the importance of lytic replication in viral pathogenesis, many processes of KSHV lytic life cycle, especially the roles of tegument proteins in virion assembly, remain poorly understood, partly due to the technical challenges inherent in studying such complex, host-related and membrane-associated processes. Through over a decade of efforts, the two collaborating groups of this application have overcome many technical hurdles and obtained a large body of preliminary data on KSHV structure and assembly. Our persistent efforts, though characterized by slow progress, has paved the road finally for rapid progress towards a thorough understanding of KHSV virion assembly. For example, the Yuan group used state-of-the-art proteomics and systems biology approaches to analyze KSHV virion protein composition and to draw a virion-wide protein interaction map. The Zhou lab has advanced cryo electron microscopy (cryoEM) and cryo electron tomography (cryoET) to visualize both ordered and pleomorphic (disordered) components of large viruses at near-atomic resolution and molecular resolution, respectively. These efforts have led to our hypotheses concerning the roles of the largest tegument protein ORF64 and a KSHV-specific tegument protein ORF45: (i) ORF64 interacts with the capsid through its C- terminal end helix bundle and connects ORF45 at its other end; (ii) tegumented KSHV capsids are transported from nuclear periphery to the trans-Golgi network (TGN) through ORF45-mediated movement along microtubules; (iiI) ORF64 guides the particles to TGN membrane where viral glycoproteins are present; and (iv) ORF45 promotes internalization of viral particles into TGN vesicles for final envelopment through a mono- ubiquitin-mediated recognition by cellular membrane sorting machinery. The proposed studies harnesses our complementary expertise to test these hypotheses. Our three specific aims are: (1) We will establish the structural basis of ORF45 and ORF64 to target lipid rafts and to bind viral glycoproteins by determining in situ tegument organization in the virion by cryoET and near-atomic resolution structure of the ordered domains ORF64 in tegumented capsids by cryoEM; (2) Guided by structure data, molecular biology approaches will be utilized to define the roles of ORF64 in virion envelopment and egress at the Golgi-derived vesicles; (3) By an integrative approach of molecular biology and 3D correlative photon/electron microscopy, we will elucidate the mechanism of ORF45-mediated targeting and internalization of viral particles into the lumen of TGN vesicles. Overall, the proposed study will yield long sought-after data on the final envelopment step and regulatory mechanisms of KSHV assembly and should inform new strategies for designing KS therapies and vaccines.